Combination therapy of a type ii anti-cd20 antibody with a selective bcl-2 inhibitor

ABSTRACT

The present invention is directed to a combination therapy involving a type II anti-CD20 antibody and a selective Bcl-2 inhibitor for the treatment of a patient suffering from cancer, particularly, a CD20-expressing cancer.

This application claims priority to U.S. Provisional No. 61/698,379,filed Sep. 7, 2012, the content of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention is directed to a combination therapy involving atype II anti-CD20 antibody and a selective Bcl-2 inhibitor for thetreatment of a patient suffering from cancer, particularly aCD20-expressing cancer.

BACKGROUND

The CD20 molecule (also called human B-lymphocyte-restricteddifferentiation antigen or Bp35) is a hydrophobic transmembrane proteinwith a molecular weight of approximately 35 kD located on pre-B andmature B lymphocytes (Valentine, M. A., et al., J. Biol. Chem. 264 (19)(1989) 11282-11287; and Einfield, D. A., et al. EMBO J. 7(3) (1988)711-717). CD20 is found on the surface of greater than 90% of B cellsfrom peripheral blood or lymphoid organs and is expressed during earlypre-B cell development and remains until plasma cell differentiation.CD20 is present on both normal B cells as well as malignant B cells. Inparticular, CD20 is expressed on greater than 90% of B cellnon-Hodgkin's lymphomas (NHL) (Anderson, K. C., et al., Blood 63(6)(1984) 1424-1433) but is not found on hematopoietic stem cells, pro-Bcells, normal plasma cells, or other normal tissues (Tedder, T. F., etal., J. Immunol. 135(2) (1985) 973-979).

The 85 amino acid carboxyl-terminal region of the CD20 protein islocated within the cytoplasm. The length of this region contrasts withthat of other B cell-specific surface structures such as IgM, IgD, andIgG heavy chains or histocompatibility antigens class I1 a or β chains,which have relatively short intracytoplasmic regions of 3, 3, 28, 15,and 16 amino acids, respectively (Komaromy, M., et al., NAR 11 (1983)6775-6785). Of the last 61 carboxyl-terminal amino acids, 21 are acidicresidues, whereas only 2 are basic, indicating that this region has astrong net negative charge. The GenBank Accession No. is NP-690605. Itis thought that CD20 might be involved in regulating an early step(s) inthe activation and differentiation process of B cells (Tedder, T. F., etal., Eur. J. Immunol. 16 (1986) 881-887) and could function as a calciumion channel (Tedder. T. F., et al., J. Cell. Biochem. 14D (1990) 195).

There exist two different types of anti-CD20 antibodies which differsignificantly in their mode of CD20 binding and biological activities(Cragg, M. S., et al., Blood 103 (2004) 2738-2743; and Cragg, M. S., etal., Blood 101 (2003) 1045-1052). Type I antibodies, as e.g. rituximab,are potent in complement mediated cytotoxicity, whereas type IIantibodies, as e.g. Tositumomab (B1), 11 B8, AT80 or humanized B-Ly 1antibodies, effectively initiate target cell death viacaspase-independent apoptosis with concomitant phosphatidylserineexposure.

The shared common features of type I and type II anti-CD20 antibodiesare summarized in Table 1 below.

TABLE 1 Properties of type I and type II anti-CD20 antibodies type Ianti-CD20 antibodies type II anti-CD20 antibodies type I CD20 epitopetype II CD20 epitope Localize CD20 to lipid rafts Do not localize CD20to lipid rafts Increased CDC (if IgG1 isotype) Decreased CDC (if IgG1isotype) ADCC activity (if IgG1 isotype) ADCC activity (if IgG1 isotype)Full binding capacity Reduced binding capacity Homotypic aggregationStronger homotypic aggregation Apoptosis induction upon cross- Strongcell death induction without linking cross-linking

The Bcl-2 family of proteins regulates programmed cell death triggeredby developmental cues and in response to multiple Stress signals (Cory.S., and Adams, J. M., Nature Reviews Cancer 2 (2002) 647-656; Adams,Genes und Development 17 (2003) 2481-2495; Danial, N. N., and Korsmeyer,S. J., Cell 116 (2004) 205-219). Whereas cell survival is promoted byBcl-2 itself and several close relatives (Bcl-xL, Bcl-W, Mcl-1 and A1),which bear three or four conserved Bcl-2 homology (BH) regions,apoptosis is driven by two other sub-families. The initial signal forcell death is conveyed by the diverse group of BH3-only proteins,including Bad, Bid, Bim, Puma and Noxa, which have in common only thesmall BH3 interaction domain (Huang and Strasser, Cell 103 (2000)839-842). However, Bax or Bak, multi-domain proteins containing BH1-BH3,are required for commitment to cell death (Cheng, et al., Molecular Cell8 (2001) 705-711; Wei, M. C., et al., Science 292 (2001) 727-730; Zong,W. X., et al., Genes and Development 15 148 (2001) 1-1486). Whenactivated, they can permeabilize the outer membrane of mitochondria andrelease pro-apoptogenic factors (e.g. cytochrome C) needed to activatethe caspases that dismantle the cell (Wang, K., Genes and Development 15(2001) 2922-2933; (Adams, 2003 supra); Green, D. R., and Kroemer, G.,Science 305 (2004) 626-629).

Interactions between members of these three factions of the Bcl-2 familydictate whether a cell lives or dies. When BH3-only proteins have beenactivated, for example, in response to DNA damage, they can bind viatheir BH3 domain to a groove on their pro-survival relatives (Sattler,et al., Science 275 (1997) 983-986). How the BH3-only and Bcl-2-likeproteins control the activation of Bax and Bak, however, remains poorlyunderstood (Adams, 2003 supra). Most attention has focused on Bax. Thissoluble monomeric protein (Hsu, Y. T., et al., Journal of BiologicalChemistry 272 (1997) 13289-1 3834; Wolter, K. G., et al., Journal ofCell Biology 139 (1997) 1281-92) normally has its membrane targetingdomain inserted into its groove, probably accounting for its cytosoliclocalization (Nechushtan, A., et al., EMBO Journal 18 (1999) 2330-2341;Suzuki, et al., Cell 103 (2000) 645-654; Schinzel, A., et al., J CellBiol 164 (2004) 1021-1032). Several unrelated peptides/proteins havebeen proposed to modulate Bax activity, reviewed in Lucken-Ardjomande,S., and Martinou, J. C., J Cell Sci 118 (2005) 473-483, but theirphysiological relevance remains to be established. Alternatively, Baxmay be activated via direct engagement by certain BH3-only proteins(Lucken-Ardjomande, S., and Martinou, J. C, 2005 supra), the bestdocumented being a truncated form of Bid, tBid (Wei, M. C., et al.,Genes und Development 14 (2000) 2060-2071; Kuwana, T., et al., Cell 111(2002) 331-342; Roucou, X., et al., Biochemical Journal 368 (2002)915-921; Cartron, P. F., et al., Mol Cell 16 (2004) 807-818). Asdiscussed elsewhere (Adams 2003 supra), the oldest model, in which Bcl-2directly engages Bax (Oltvai, Z. N., et al., Cell 74 (1993) 609-619),has become problematic because Bcl-2 is membrane bound while Bax iscytosolic, and their interaction seems highly dependent on thedetergents used for cell lysis (Hsu, Y. T., and Youle, 1997 supra).Nevertheless, it is well established that the BH3 region of Bax canmediate association with Bcl-2 (Zha, H., and Reed, J., Journal ofBiological Chemistry 272 (1997) 31482-88; Wang, K., et al., Molecularund Cellular Biology 18 (1998) 6083-6089) and that Bcl-2 prevents theoligomerization of Bax, even though no heterodimers can be detected(Mikhailov, V., et al., Journal of Biological Chemistry 276 (2001)18361-18374). Thus, whether the pro-survival proteins restrain Baxactivation directly or indirectly remains uncertain.

Although Bax and Bak seem in most circumstances to be functionallyequivalent (Lindsten, T., et al., Molecular Cell 6 (2000) 1389-1399;Wei, M. C., et al., 2001 supra), substantial differences in theirregulation would be expected from their distinct localization in healthycells. Unlike Bax, which is largely cytosolic, Bak resides in complexeson the outer membrane of mitochondria and on the endoplasmic reticulumof healthy cells (Wei, M. C., et al., 2000 supra; Zong, W. X., et al.,Journal of Cell Biology 162 (2003) 59-69). Nevertheless, on receipt ofcytotoxic signals, both Bax and Bak change conformation, and Baxtranslocates to the organellar membranes, where both Bax and Bak thenform homo-oligomers that can associate, leading to membranepermeabilization (Hsu. Y. T., et al., PNAS 94 (1997) 3668-3672; Wolter,K. G., et al., 1997 supra; Antonsson, B., et al., Journal of BiologicalChemistry 276 (2001) 11615-11623; Nechushtan, A., et al., Journal ofCell Biology 153 (2001) 1265-1276; Wei, M. C., et al., 2001 supra;Mikhailov, V., et al., Journal of Biological Chemistry 278 (2003)5367-5376).

There exist various Bcl-2 inhibitors, which all have the same propertyof inhibiting prosurvival members of the Bcl-2 family of proteins andare therefore promising candidates for the treatment of cancer. SuchBcl-2 inhibitors are e.g. Oblimersen, SPC-2996, RTA-402, Gossypol,AT-101, Obatoclax mesylate, A-371191, A-385358, A-438744, ABT-737,ABT-263, AT-101, BL-11, BL-193, GX-15-003, 2-Methoxyantimycin A₃,HA-14-1, KF-67544, Purpurogallin, TP-TW-37, YC-137 and Z-24, and aredescribed e.g. in Zhai, D., et al., Cell Death and Differentiation 13(2006) 1419-1421.

Smith, M. R., et al, Molecular Cancer Therapeutics 3(12) (2004)1693-1699 and Ramanarayanan, J. et al., British Journal of Haematology127(5) (2004) 519-530, refer to a combination of a type I anti-CD20antibody (rituximab) with antisense Bcl-2 oligonucleotides (Oblimersen).

SUMMARY OF THE INVENTION

Provided herein are methods for the treatment of a patient sufferingfrom cancer, comprising co-administering, to a patient in need of suchtreatment, a type II anti-CD20 antibody and a selective Bcl-2 inhibitor.The co-administration may be simultaneous or sequential in either order.

An example of the type II anti-CD20 antibody for use in the presentinvention is a GA101 antibody.

In an embodiment, the type II anti-CD20 antibody has increased antibodydependent cellular cytotoxicity (ADCC).

In an embodiment, at least 40% of the oligosaccharides of the Fc regionof said type II anti-CD20 antibody are non-fucosylated.

In an embodiment, the selective Bcl-2 inhibitor is GDC-0199 (also knownas ABT-199), or a pharmaceutically acceptable salt thereof.

In an embodiment, the cancer is a non-solid tumor.

In certain embodiments, methods are provided for the treatment of acancer in a human in need thereof comprising administering to said humana GA101 antibody and/or GDC-0199 in multiple dosing cycles. In anembodiment, each dosing cycle of the multiple dosing cycle is for atleast 1 week. In an embodiment, each dosing cycle of the multiple dosingcycle is for at 2, for at least 3, for at least 4, for at least 5, orfor at least 6 weeks.

In an embodiment wherein the GA101 antibody and GDC-0199 areadministered to the human in multiple dosing cycles, GA101 antibody can,for example, be administered once per dosing cycle for one or moredosing cycles of the multiple dosing cycles. The amount of GA101administered per dose can, for example, be between about 300 mg to about3000 mg, or between about 500 mg to about 3000 mg, or about 500 mg toabout 1200 mg.

In an embodiment wherein the GA101 antibody and GDC-0199 areadministered to the human in multiple dosing cycles, GDC-0199 can, forexample, be administered each day in a dosing cycle for one or moredosing cycles of the multiple dosing cycles. In an embodiment, GDC-0199is administered in fewer than all of the days of the initial dosingcycle, and is administered each day of the dosing cycles of the multipledosing cycles that follow the initial dosing cycle. The amount ofGDC-0199 administered per day can be between about 10 mg to about 1,000mg, about 20 mg to about 800 mg, about 20 mg to about 500 mg, or betweenabout 50 mg to about 300 mg.

In an embodiment, both the GA101 antibody and GDC-0199 are administeredto the patient in at least 2, 3, 4, 5, 6, 7, 8, or more than 8, dosingcycles of the multiple dosing cycles.

In certain embodiments of the methods provided for the treatment of acancer in a human in need thereof comprising administering to said humanboth a GA101 antibody and GDC-0199 in multiple dosing cycles, followingthe last dosing cycle of multiple dosing cycles, GDC-0199 alone can beadministered to the human in the absence of the GA101 antibody, or theGA101 antibody alone can be administered to the patient in the absenceof GDC-0199. For instance, when GDC-0199 is administered alone to thehuman (e.g., following the last cycle of multiple dosing cycles whereinboth GDC-0199 and the GA101 antibody are administered to the human),GDC-0199 can be administered to the human for at least 3, 4, 5, 6, 7, 8or 9 days, or for 10 or more days, for 20 or more days, or for 30 ormore days.

In yet another embodiment of the methods provided wherein a GA101antibody and GDC-0199 are administered to the patient in multiple dosingcycles, the multiple dosing cycles comprise an escalating dosing cyclein which GDC-0199 is administered to the patient in escalating dailydose amounts during the escalating dosing cycle.

DESCRIPTION OF THE FIGURES

FIG. 1 . Antitumor activity of combined treatment of a type II anti-CD20antibody (GA101 antibody, in this case, obinutuzumab) with a Bcl-2inhibitor (ABT-199, a.k.a. GDC-0199). Arrows and line under the x-axisindicates the days of dosing of GA101 and GDC-0199, respectively.

FIG. 2 . Exemplary dosing schedule for administering GDC-199 withobinutuzumab.

FIG. 3 . Exemplary dosing schedule for administering GDC-199 withobinutuzumab.

FIG. 4 . Antitumor activities of a type II anti-CD20 antibody(obinutuzumab, a.k.a. RO5072759) used alone or in combination withGDC-0199, and of a type I anti-CD20 antibody (rituximab) used alone orin combination with GDC-0199 on human Z138 mantle cell lymphoma cells.

FIG. 5 . Results from xenograft model of aggressive lymphomademonstrating that single agent treatment with GDC-0199 followingcombination of GDC-0199 with type II anti-CD20 antibody (GA101 antibody,in this case, obinutuzumab) delays tumor regrowth.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the method described above.

The present invention also relates to a method for the treatment of ahuman in need thereof comprising administering to said human aneffective amount of a GA101 antibody or2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods, followed by co-administering an effective amount of said GA101antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods.

The present invention also relates to a method for the treatment of ahuman in need thereof comprising administering to said human aneffective amount of a GA101 antibody or2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamidefor 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, followedby co-administering an effective amount of said GA101 antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods.

The present invention also relates to a method for the treatment of ahuman in need thereof comprising administering an effective amount ofsaid GA101 antibody for 1, 2, 3, 4, 5, 6, or 7 days, followed byco-administering an effective amount of said GA101 antibody antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods.

The present invention also relates to a method for the treatment of ahuman in need thereof comprising administering an effective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for 1, 2, 3, 4, 5, 6, or 7days, followed by co-administering an effective amount of said GA101antibody antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods.

The present invention also relates to a method for the treatment of ahuman in need thereof comprising administering an effective amount ofsaid GA101 antibody once every dosing period for 1, 2, 3, 4, 5 or 6cycles, followed by co-administering an effective amount of said GA101antibody antibody once every dosing period and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof one, two or three times aday for one or more dosing periods.

The present invention also relates to a method for the treatment of ahuman in need thereof comprising administering an effective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof one, two or three times aday for 1, 2, 3, 4, 5 or 6 dosing periods, followed by co-administeringan effective amount of said GA101 antibody once every dosing period and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof one, two or three times aday for one or more dosing periods.

The present invention also relates to any one of the above methods,wherein the effective amount of said GA101 antibody is 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 mgand the effective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, or 1000 mg.

The present invention also relates to any one of the above methods,wherein the effective amount of said GA101 antibody is 800, 900, 1000,1100, 1200, 1300, 1400 or 1500 mg, and the effective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290 or 300 mg.

The present invention also relates to any one of the above methods,wherein when said cancer is NHL, the effective amount of said GA101antibody is 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 mg, and theeffective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,and 800 mg.

The present invention also relates to any one of the above methods,wherein when said cancer is AML, the effective amount of said GA101antibody is 800.900, 1000, 1100, 1200, 1300, 1400 or 1500 mg, and theeffective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,and 800 mg.

The present invention also relates to any one of the above methods,wherein said GA101 antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof were co-administeredsequentially during each dosing period, and each dosing period is 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 days.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

In one embodiment, said type II anti-CD20 antibody is a monoclonalantibody.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred. Such murine/human chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding murine immunoglobulin variable regions and DNAsegments encoding human immunoglobulin constant regions. Other forms of“chimeric antibodies” encompassed by the present invention are those inwhich the class or subclass has been modified or changed from that ofthe original antibody. Such “chimeric” antibodies are also referred toas “class-switched antibodies.” Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques now well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann. L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric and bifunctional antibodies.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. Human antibodies are well-known inthe state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr.Opin. Pharmacol. 5 (2001) 368-374). Based on such technology, humanantibodies against a great variety of targets can be produced. Examplesof human antibodies are for example described in Kellermann, S. A., etal., Curr Opin Biotechnol. 13 (2002) 593-597.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences in a rearranged form. Therecombinant human antibodies according to the invention have beensubjected to in vivo somatic hypermutation. Thus, the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH andVL sequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

As used herein, “specifically binding” or “binds specifically to” refersa binding that is sufficiently selective to a target as to distinguishit from a binding tounwanted or nonspecific targets (e.g., an antibodythat specifically binds to a human CD20). In one embodiment, a GA101antibody of this invention has a binding affinity for human CD20 (Kd) of≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g.10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³M). In yet another embodiment, the KD is 10⁻¹⁰ mol/l or lower (e.g.10⁻¹² mol/l). The binding affinity is determined with a standard bindingassay, such as Scatchard plot analysis on CD20 expressing cells.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded. In one embodiment, it isdouble-stranded DNA.

The “constant domains” are not involved directly in binding the antibodyto an antigen but are involved in the effector functions (ADCC,complement binding, and CDC).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W. H. Freeman and Co., page 91(2007).)

The term “hypervariable region” or “—HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, 1H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (112), and 93-101 (H3) (MacCallumet al. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a). (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Synonyms of CD20, as recognized in the art, include B-lymphocyte antigenCD20, B-lymphocyte surface antigen B1. Leu-16, Bp35, BM5, and LF5.

The term “anti-CD20 antibody” according to the invention is an antibodythat binds specifically to CD20 antigen. Depending on binding propertiesand biological activities of anti-CD20 antibodies to the CD20 antigen,two types of anti-CD20 antibodies (type I and type II anti-CD20antibodies) can be distinguished according to Cragg, M. S., et al.,Blood 103 (2004) 2738-2743; and Cragg, M. S., et al., Blood 101 (2003)1045-1052, see Table 2.

TABLE 2 Properties of type I and type II anti-CD20 antibodies type Ianti-CD20 antibodies type II anti-CD20 antibodies type I CD20 epitopetype II CD20 epitope Localize CD20 to lipid rafts Do not localize CD20to lipid rafts Increased CDC (if IgG1 isotype) Decreased CDC (if IgG1isotype) ADCC activity (if IgG1 isotype) ADCC activity (if IgG1 isotype)Full binding capacity Reduced binding capacity Homotypic aggregationStronger homotypic aggregation Apoptosis induction upon cross- Strongcell death induction linking without cross-linking

One property of type I and type II anti-CD20 antibodies is their mode ofbinding. Type I and type II anti-CD20 antibodies can be classified bythe ratio of the binding capacities to CD20 on Raji cells (ATCC-No.CCL-86) of said anti-CD20 antibody compared to rituximab.

The type II anti-CD20 antibodies have a ratio of the binding capacitiesto CD20 on Raji cells (ATCC-No. CCL-86) of said anti-CD20 antibodycompared to rituximab of 0.3 to 0.6, and in one embodiment of 0.35 to0.55, and in another embodiment, 0.4 to 0.5. Examples of such type IIanti-CD20 antibodies include e.g. tositumomab (B1 IgG2a), GA101 antibodyIgG1 (a chimeric humanized IgG1 antibody as disclosed in WO2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1.In one embodiment, said type II anti-CD20 antibody is a monoclonalantibody that binds to the same epitope as GA101 antibody (as disclosedin WO 2005/044859).

The “ratio of the binding capacities to CD20 on Raji cells (ATCC-No.CCL-86) of an anti-CD20 antibodies compared to rituximab” is determinedby direct immunofluorescence measurement (the mean fluorescenceintensities (MFI) is measured) using said anti-CD20 antibody conjugatedwith Cy5 and rituximab conjugated with Cy5 in a FACSArray (BectonDickinson) with Raji cells (ATCC-No. CCL-86), as described in ExampleNo. 2, and calculated as follows:

${{Ratio}{of}{the}{binding}{capacities}{}{to}{CD}20{on}{Raji}{cells}\left( {{ATCC}‐{{No}.{CCL}}‐86} \right)} = {\frac{{MFI}\left( {{{Cy}5}‐{anti}‐{{CD}20{antibody}}} \right)}{{MFI}\left( {{{Cy}5}‐{rituximab}} \right)} \times \frac{{{Cy}5}‐{{labeling}{{ratio}\left( {{{Cy}5}‐{rituximab}} \right)}}}{{{Cy}5}‐{{labeling}{{ratio}\left( {{{Cy}5}‐{anti}‐{{CD}20{antibody}}} \right)}}}}$

MFI is the mean fluorescent intensity. The “Cy5-labeling ratio” as usedherein means the number of Cy5-label molecules per molecule antibody.

Typically said type II anti-CD20 antibody has a ratio of the bindingcapacities to CD20 on Raji cells (ATCC-No. CCL-86) of said secondanti-CD20 antibody compared to rituximab of 0.3 to 0.6, and in oneembodiment, 0.35 to 0.55, and in yet another embodiment, 0.4 to 0.5.

In one embodiment said type II anti-CD20 antibody, e.g., a GA101antibody, has increased antibody dependent cellular cytotoxicity (ADCC).

By “antibody having increased antibody dependent cellular cytotoxicity(ADCC)”, it is meant an antibody, as that term is defined herein, havingincreased ADCC as determined by any suitable method known to those ofordinary skill in the art. One accepted in vitro ADCC assay is asfollows:

-   -   1) the assay uses target cells that are known to express the        target antigen recognized by the antigen-binding region of the        antibody;    -   2) the assay uses human peripheral blood mononuclear cells        (PBMCs), isolated from blood of a randomly chosen healthy donor,        as effector cells;    -   3) the assay is carried out according to following protocol:        -   i) the PBMCs are isolated using standard density            centrifugation procedures and are suspended at 5×10⁶            cells/ml in RPMI cell culture medium;        -   ii) the target cells are grown by standard tissue culture            methods, harvested from the exponential growth phase with a            viability higher than 90%, washed in RPMI cell culture            medium, labeled with 100 micro-Curies of ⁵¹Cr, washed twice            with cell culture medium, and resuspended in cell culture            medium at a density of 10⁵ cells/ml;        -   iii) 100 microliters of the final target cell suspension            above are transferred to each well of a 96-well microtiter            plate;        -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04            ng/ml in cell culture medium and 50 microliters of the            resulting antibody solutions are added to the target cells            in the 96-well microtiter plate, testing in triplicate            various antibody concentrations covering the whole            concentration range above;        -   v) for the maximum release (MR) controls, 3 additional wells            in the plate containing the labeled target cells, receive 50            microliters of a 2% (VN) aqueous solution of non-ionic            detergent (Nonidet, Sigma, St. Louis), instead of the            antibody solution (point iv above);        -   vi) for the spontaneous release (SR) controls, 3 additional            wells in the plate containing the labeled target cells,            receive 50 microliters of RPMI cell culture medium instead            of the antibody solution (point iv above);        -   vii) the 96-well microtiter plate is then centrifuged at            50×g for 1 minute and incubated for 1 hour at 4° C.;        -   viii) 50 microliters of the PBMC suspension (point i above)            are added to each well to yield an effector:target cell            ratio of 25:1 and the plates are placed in an incubator            under 5% CO2 atmosphere at 37° C. for 4 hours;        -   ix) the cell-free supernatant from each well is harvested            and the experimentally released radioactivity (ER) is            quantified using a gamma counter;        -   x) the percentage of specific lysis is calculated for each            antibody concentration according to the formula            (ER-MR)/(MR-SR)×100, where ER is the average radioactivity            quantified (see point ix above) for that antibody            concentration, MR is the average radioactivity quantified            (see point ix above) for the MR controls (see point V            above), and SR is the average radioactivity quantified (see            point ix above) for the SR controls (see point vi above):    -   4) “increased ADCC” is defined as either an increase in the        maximum percentage of specific lysis observed within the        antibody concentration range tested above, and/or a reduction in        the concentration of antibody required to achieve one half of        the maximum percentage of specific lysis observed within the        antibody concentration range tested above. In one embodiment,        the increase in ADCC is relative to the ADCC, measured with the        above assay, mediated by the same antibody, produced by the same        type of host cells, using the same standard production,        purification, formulation and storage methods, which are known        to those skilled in the art, except that the comparator antibody        (lacking increased ADCC) has not been produced by host cells        engineered to overexpress GnTIII and/or engineered to have        reduced expression from the fucosyltransferase 8 (FUT8) gene        (e.g., including, engineered for FUT8 knock out).

Said “increased ADCC” can be obtained by, for example, mutating and/orglycoengineering of said antibodies. In one embodiment, the antibody isglycoengineered to have a biantennary oligosaccharide attached to the Feregion of the antibody that is bisected by GIcNAc, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); US 2005/0123546 (Umana et al.), Umana, P., et al., NatureBiotechnol. 17 (1999) 176-180). In another embodiment, the antibody isglycoengineered to lack fucose on the carbohydrate attached to the Fcregion by expressing the antibody in a host cell that is deficient inprotein fucosylation (e.g., Lec13 CHO cells or cells having analpha-1,6-fucosyltransferase gene (FUT8) deleted or the FUT geneexpression knocked down (see, e.g., Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng.,94(4):680-688 (2006); and WO2003/085107). In yet another embodiment, theantibody sequence has been engineered in its Fc region to enhance ADCC(e.g., in one embodiment, such engineered antibody variant comprises anFc region with one or more amino acid substitutions at positions 298,333, and/or 334 of the Fc region (EU numbering of residues)).

The term “complement-dependent cytotoxicity (CDC)” refers to lysis ofhuman tumor target cells by the antibody according to the invention inthe presence of complement. CDC can be measured by the treatment of apreparation of CD20 expressing cells with an anti-CD20 antibodyaccording to the invention in the presence of complement. CDC is foundif the antibody induces at a concentration of 100 nM the lysis (celldeath) of 20% or more of the tumor cells after 4 hours. In oneembodiment, the assay is performed with ⁵¹Cr or Eu labeled tumor cellsand measurement of released ⁵¹Cr or Eu. Controls include the incubationof the tumor target cells with complement but without the antibody.

The term “GA101 antibody” as used herein refers to any one of thefollowing antibodies that bind human CD20: (1) an antibody comprising anHVR-H1 comprising the amino acid sequence of SEQ ID NO:1, an HVR-H2comprising the amino acid sequence of SEQ ID NO:2, an HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:3, an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:4, an HVR-L2 comprising the amino acidsequence of SEQ ID NO:5, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:6; (2) an antibody comprising a VH domaincomprising the amino acid sequence of SEQ ID NO:7 and a VL domaincomprising the amino acid sequence of SEQ ID NO:8, (3) an antibodycomprising an amino acid sequence of SEQ ID NO:9 and an amino acidsequence of SEQ ID NO: 10; (4) an antibody known as obinutuzumab, or (5)an antibody that comprises an amino acid sequence that has at least 95%,96%, 97%, 98% or 99% sequence identity with amino acid sequence of SEQID NO:9 and that comprises an amino acid sequence that has at least 95%,96%, 97%, 98% or 99% sequence identity with an amino acid sequence ofSEQ ID NO: 10. In one embodiment, the GA101 antibody is an IgG1 isotypeantibody

The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein function can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions. (Jenkins, N., et al.,Nature Biotechnol. 14 (1996) 975-981).

Mammalian cells are the preferred hosts for production of therapeuticglycoproteins, due to their capability to glycosylate proteins in themost compatible form for human application. (Cumming, D. A., et al.,Glycobiology 1 (1991) 115-130; Jenkins, N., et al., Nature Biotechnol.14 (1996) 975-981). Bacteria very rarely glycosylate proteins, and likeother types of common hosts, such as yeasts, filamentous fungi, insectand plant cells, yield glycosylation patterns associated with rapidclearance from the blood stream, undesirable immune interactions, and insome specific cases, reduced biological activity. Among mammalian cells,Chinese hamster ovary (CHO) cells have been most commonly used duringthe last two decades. In addition to giving suitable glycosylationpatterns, these cells allow consistent generation of genetically stable,highly productive clonal cell lines. They can be cultured to highdensities in simple bioreactors using serum free media, and permit thedevelopment of safe and reproducible bioprocesses. Other commonly usedanimal cells include baby hamster kidney (BHK) cells, NSO- andSP2/0-mouse myeloma cells. More recently, production from transgenicanimals has also been tested. (Jenkins, N., et al., Nature Biotechnol.14 (1996) 975-981).

All antibodies contain carbohydrate structures at conserved positions inthe heavy chain constant regions, with each isotype possessing adistinct array of N-linked carbohydrate structures, which variablyaffect protein assembly, secretion or functional activity. (Wright, A.,and Monison, S. L., Trends Biotech. 15 (1997) 26-32). The structure ofthe attached N-linked carbohydrate varies considerably, depending on thedegree of processing, and can include high-mannose, multiply-branched aswell as biantennary complex oligosaccharides. (Wright. A., and Morrison,S. L., Trends Biotech. 15 (1997) 26-32). Typically, there isheterogeneous processing of the core oligosaccharide structures attachedat a particular glycosylation site such that even monoclonal antibodiesexist as multiple glycoforms. Likewise, it has been shown that majordifferences in antibody glycosylation occur between cell lines, and evenminor differences are seen for a given cell line grown under differentculture conditions. (Lifely, M. R., et al., Glycobiology 5 (1995)813-822).

One way to obtain large increases in potency, while maintaining a simpleproduction process and potentially avoiding significant, undesirableside effects, is to enhance the natural, cell-mediated effectorfunctions of monoclonal antibodies by engineering their oligosaccharidecomponent as described in Umana. P., et al., Nature Biotechnol. 17(1999) 176-180 and U.S. Pat. No. 6,602,684. IgG1 type antibodies, themost commonly used antibodies in cancer immunotherapy, are glycoproteinsthat have a conserved N-linked glycosylation site at Asn297 in each CH2domain. The two complex biantennary oligosaccharides attached to Asn297are buried between the CH2 domains, forming extensive contacts with thepolypeptide backbone, and their presence is essential for the antibodyto mediate effector functions such as antibody dependent cellularcytotoxicity (ADCC) (Lifely, M. R., et al., Glycobiology 5 (1995)813-822; Jefferis, R., et al., Immunol. Rev. 163 (1998) 59-76; Wright,A., and Morrison, S. L., Trends Biotechnol. 15 (1997) 26-32).

It was previously shown that overexpression in Chinese hamster ovary(CHO) cells of β(1,4)-N-acetylglucosaminyltransferase III (“GnTIII), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofan antineuroblastoma chimeric monoclonal antibody (chCE7) produced bythe engineered CHO cells. (See Umana, P., et al., Nature Biotechnol. 17(1999) 176-180; and WO 99/154342, the entire contents of which arehereby incorporated by reference). The antibody chCE7 belongs to a largeclass of unconjugated monoclonal antibodies which have high tumoraffinity and specificity, but have too little potency to be clinicallyuseful when produced in standard industrial cell lines lacking theGnTIII enzyme (Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180).That study was the first to show that large increases of ADCC activitycould be obtained by engineering the antibody producing cells to expressGnTIII, which also led to an increase in the proportion of constantregion (Fc)-associated, bisected oligosaccharides, including bisected,non-fucosylated oligosaccharides, above the levels found innaturally-occurring antibodies.

In one embodiment, a composition comprising a GA101 antibody of thisinvention comprises GA101 antibodies engineered to have increased ADCCactivity.

The term “Bcl-2” as used herein refers to the Bcl-2 protein (Swiss ProtID No. P10415), a member of the Bcl-2 family of proteins (Cory, S., andAdams, J. M., Nature Reviews Cancer 2 (2002) 647-656; Adams, Genes undDevelopment 17 (2003) 2481-2495; Danial, N. N., and Korsmeyer, S. J.,Cell 116 (2004) 205-219; Petros, A. M., Biochim Biophys Acta 1644 (2004)83-94).

The term “selective Bcl-2 inhibitors” as used herein refers to2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamide,(a.k.a.ABT-199 or GDC-0199), a Bcl-2 inhibitor of formula I, which is describedin International Publication No. WO2010/138588 and in US publication NO.US2010/0305122, which are incorporated by reference herein.

The term “expression of the CD20” antigen is intended to indicate ansignificant level of expression of the CD20 antigen in a cell, e.g., aT- or B-Cell. In one embodiment, patients to be treated according ot themethods of this invention express significant levels of CD20 on a B-celltumor or cancer. Patients having a “CD20 expressing cancer” can bedetermined by standard assays known in the art. e.g., CD20 antigenexpression is measured using immunohistochemical (IHC) detection, FACSor via PCR-based detection of the corresponding mRNA.

The term “CD20 expressing cancer” as used herein refers to all cancersin which the cancer cells show an expression of the CD20 antigen. SuchCD20 expressing cancer may be, for example, lymphomas, lymphocyticleukemias, lung cancer, non small cell lung (NSCL) cancer,bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer,skin cancer, cancer of the head or neck, cutaneous or intraocularmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, gastric cancer, colon cancer, breastcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, prostate cancer, cancer of the bladder, cancer of the kidneyor ureter, renal cell carcinoma, carcinoma of the renal pelvis,mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of thecentral nervous system (CNS), spinal axis tumors, brain stem glioma,glioblastoma multiforme, astrocytomas, schwanomas, ependymonas,medulloblastomas, meningiomas, squamous cell carcinomas, pituitaryadenoma, including refractory versions of any of the above cancers, or acombination of one or more of the above cancers.

In one embodiment, CD20 expressing cancer as used herein refers tolymphomas (e.g., B-Cell Non-Hodgkin's lymphomas (NHL)) and lymphocyticleukemias. Such lymphomas and lymphocytic leukemias include e.g. a)follicular lymphomas, b) Small Non-Cleaved Cell Lymphomas/Burkitt'slymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt'slymphoma and Non-Burkitt's lymphoma) c) marginal zone lymphomas(including extranodal marginal zone B cell lymphoma (Mucosa-associatedlymphatic tissue lymphomas. MALT), nodal marginal zone B cell lymphomaand splenic marginal zone lymphoma), d) Mantle cell lymphoma (MCL), e)Large Cell Lymphoma (including B-cell diffuse large cell lymphoma(DLCL), Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma, PrimaryMediastinal B-Cell Lymphoma, Angiocentric Lymphoma-Pulmonary B-CellLymphoma) f) hairy cell leukemia, g) lymphocytic lymphoma, waldenstrom'smacroglobulinemia, h) acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cellprolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma,multiple myeloma, plasmacytoma, j) Hodgkin's disease, k) acute myeloidleukemia (AML); among other types of lymphomas and lymphocycticluekemias.

In one embodiment, the CD20 expressing cancer is a B-Cell Non-Hodgkin'slymphomas (NHL). In another embodiment, the CD20 expressing cancer is aMantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL),acute myeloid leukemia (AML). Burkitt's lymphoma, hairy cell leukemia,follicular lymphoma, multiple myeloma, marginal zone lymphoma, posttransplant lymphoproliferative disorder (PTLD), HIV associated lymphoma,waldenstrom's macroglobulinemia, or primary CNS lymphoma.

“Relapsed or Refractory” CLL as used herein includes CLL patients whohave received at least 1 prior chemotherapy containing treatmentregimen. Relapsed patients generally have developed progressive diseasefollowing a response to the prior chemotherapy-containing treatmentregimen. Refractory patients have generally failed to respond orrelapsed within 6 months to the last prior chemotherapy-containingregimen.

“Previously untreated” CLL as used herein includes patients diagnosedwith CLL, but who have, in general, received no prior chemotherapy orimmunotherapy. Patients with a history of emergency, loco-regionalradiotherapy (e.g., for relief of compressive signs or symptoms) orcorticosteroids can still be considered previously untreated.

The term “treating” as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing,either partially or completely, the growth of tumors, tumor metastases,or other cancer-causing or neoplastic cells in a patient. The term“treatment” as used herein, unless otherwise indicated, refers to theact of treating.

The term “a method of treating” or its equivalent, when applied to, forexample, cancer refers to a procedure or course of action that isdesigned to reduce or eliminate the number of cancer cells in a patient,or to alleviate the symptoms of a cancer. “A method of treating” canceror another proliferative disorder does not necessarily mean that thecancer cells or other disorder will, in fact, be eliminated, that thenumber of cells or disorder will, in fact, be reduced, or that thesymptoms of a cancer or other disorder will, in fact, be alleviated.Often, a method of treating cancer will be performed even with a lowlikelihood of success, but which, given the medical history andestimated survival expectancy of a patient, is nevertheless deemed toinduce an overall beneficial course of action. The terms“co-administration” or “co-administering” refer to the administration ofsaid type II anti-CD20 antibody and said selective Bcl-2 inhibitor astwo separate formulations. The co-administration can be simultaneous orsequential in either order. In one further embodiment, there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. Said type II anti-CD20 antibody and saidselective Bcl-2 inhibitor are co-administered either simultaneously orsequentially (e.g. via an intravenous (i.v.) through a continuousinfusion (one for the antibody and eventually one for the Bcl-2inhibitor; or the Bcl-2 inhibitor is administered orally). When boththerapeutic agents are co-administered sequentially the agents areadministered in two separate administrations that are separated by a“specific period of time”. The term specific period of time is meant anywhere from 1 hour to 15 days. For example, one of the agents can beadministered within about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 day, or 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour from the administration of theother agent, and, in one embodiment, the specific period time is 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 day, or 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour.

The term “simultaneously” means at the same time or within a shortperiod of time, usually less than 1 hour.

A dosing period as used herein is meant a period of time, during whicheach therapeutic agent has been administered at least once. A dosingcycle is usually about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days,and, in one embodiment, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, forexample, 7 or 14 days.

In certain embodiments, a dosing period is a dosing cycle.

It is self-evident that the antibodies are administered to the patientin a “therapeutically effective amount” (or simply “effective amount”)which is the amount of the respective compound or combination that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician. The administration of an effective amount ofa therapeutically agent can be a single administration or split doseadministration. “split dose administration” is meant an effective amountis a split into multiple doses, preferably 2, and administered within 1or 2 days. For example, if 100 mg of a selective BCL-2 inhibitor isdeemed effective, it can be administered in one 100 mg administration ortwo 50 mg administrations. Split dose administration is sometimesdesirable at the beginning of a dosing period to reduce side effects.When an effective amount is administered in split dosing, it is stillconsidered one administration of an effective amount. For example, when100 mg is the effective amount of a selective Bcl-2 inhibitor and thatamount is administered in two 50 mg doses over a period of time, e.g. 2days, only one effective amount is administered during that period oftime.

The amount of co-administration of said type II anti-CD20 antibody andsaid Bcl-2 inhibitor and the timing of co-administration will depend onthe type (species, gender, age, weight, etc.) and condition of thepatient being treated and the severity of the disease or condition beingtreated. Said type II anti-CD20 antibody and said Bcl-2 inhibitor aresuitably co-administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of said type II anti-CD20antibody, and 0.1 mg/kg to 200 mg/kg (e.g. 10-150 mg/kg) of saidselective Bcl-2 inhibitor is an initial candidate dosage forco-administration of both drugs to the patient. If the administration isintravenous the initial infusion time for said type II anti-CD20antibody or said Bcl-2 inhibitor may be longer than subsequent infusiontimes, for instance approximately 90 minutes for the initial infusion,and approximately 30 minutes for subsequent infusions (if the initialinfusion is well tolerated).

In one embodiment, the preferred dosage of said type II anti-CD20antibody will be in the range from about 0.05 mg/kg to about 30 mg/kg,preferably 1 mg/kg to 30 mg/kg; or 500 mg-3000 mg flat dose. Thus, oneor more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 10 mg/kg or 30mg/kg or 500 mg-3000 mg flat dose (or any combination thereof) may beco-administered to the patient. The preferred dosage of said Bcl-2inhibitor will be in the range from 20 mg/kg to about 150 mg/kg,preferably 1 mg/kg to 10 mg/kg. Depending on the on the type (species,gender, age, weight, etc.) and condition of the patient and on the typeof anti-CD20 antibody and Bcl-2 inhibitor, the dosage and theadministration schedule of said anti-CD20 antibody can differ from thedosage of Bcl-2 inhibitor. E.g. the said anti-CD20 antibody may beadministered e.g. every one to three weeks and said Bcl-2 inhibitor maybe administered daily or every 2 to 7 days. An initial higher loadingdose, followed by one or more lower doses may also be administered.

The present invention relates in part to a composition comprising a typeII anti-CD20 antibody and a selective Bcl-2 inhibitora selective Bcl-2inhibitor.

In a preferred embodiment, the composition of the present invention isuseful for preventing or reducing metastasis or further dissemination insuch a patient suffering from CD20 expressing cancer. The composition isuseful for increasing the duration of survival of such a patient,increasing the progression free survival of such a patient, increasingthe duration of response, resulting in a statistically significant andclinically meaningful improvement of the treated patient as measured bythe duration of survival, progression free survival, response rate orduration of response. In a preferred embodiment, the composition isuseful for increasing the response rate in a group of patients.

In the context of this invention, additional other cytotoxic,chemotherapeutic or anti-cancer agents, or compounds that enhance theeffects of such agents (e.g. cytokines) may be used in the type IIanti-CD20 antibody and Bcl-2 inhibitor combination treatment of CD20expressing cancer. Such molecules are suitably present in combination inamounts that are effective for the purpose intended. Preferably the typeII anti-CD20 antibody and Bcl-2 inhibitor combination treatment is usedwithout such additional cytotoxic, chemotherapeutic or anti-canceragents, or compounds that enhance the effects of such agents.

Such agents include, for example: alkylating agents or agents with analkylating action, such as cyclophosphamide (CTX; e.g. CYTOXAN®),chlorambucil (CHL; e.g. LEUKERAN®), cisplatin (CisP; e.g. PLATINOL®)busulfan (e.g. MYLERAN®), melphalan, carmustine (BCNU), streptozotocin,triethylenemelamine (TEM), mitomycin C, and the like; anti-metabolites,such as methotrexate (MTX), etoposide (VP16; e.g. VEPESID®),6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C),5-fluorouracil (5-FU), capecitabine (e.g. XELODA®), dacarbazine (DTIC),and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g.ADRIAMYCIN®), daunorubicin (daunomycin), bleomycin, mithramycin and thelike; alkaloids, such as vinca alkaloids such as vincristine (VCR),vinblastine, and the like; and other antitumor agents, such aspaclitaxel (e.g. TAXOL®) and paclitaxel derivatives, the cytostaticagents, glucocorticoids such as dexamethasone (DEX; e.g. DECADRON®) andcorticosteroids such as prednisone, nucleoside enzyme inhibitors such ashydroxyurea, amino acid depleting enzymes such as asparaginase,leucovorin and other folic acid derivatives, and similar, diverseantitumor agents. The following agents may also be used as additionalagents: arnifostine (e.g. ETHYOL®), dactinomycin, mechlorethamine(nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU),doxorubicin lipo (e.g. DOXIL®), gemcitabine (e.g. GEMZAR®), daunorubicinlipo (e.g. DAUNOXOME®), procarbazine, mitomycin, docetaxel (e.g.TAXOTERE®), aldesleukin, carboplatin, oxaliplatin, cladribine,camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin(SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna,interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide,megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen,teniposide, testolactone, thioguanine, thiotepa, uracil mustard,vinorelbine, chlorambucil. Preferably the type II anti-CD20 antibody andBcl-2 inhibitor combination treatment is used without such additionalagents.

The use of the cytotoxic and anticancer agents described above as wellas antiproliferative target-specific anticancer drugs like proteinkinase inhibitors in chemotherapeutic regimens is generally wellcharacterized in the cancer therapy arts, and their use herein fallsunder the same considerations for monitoring tolerance and effectivenessand for controlling administration routes and dosages, with someadjustments. For example, the actual dosages of the cytotoxic agents mayvary depending upon the patient's cultured cell response determined byusing histoculture methods. Generally, the dosage will be reducedcompared to the amount used in the absence of additional other agents.

Typical dosages of an effective cytotoxic agent can be in the rangesrecommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

In the context of this invention, an effective amount of ionizingradiation may be carried out and/or a radiopharmaceutical may be used inaddition to the type II anti-CD20 antibody and Bcl-2 inhibitorcombination treatment of CD20 expressing cancer. The source of radiationcan be either external or internal to the patient being treated. Whenthe source is external to the patient, the therapy is known as externalbeam radiation therapy (EBRT). When the source of radiation is internalto the patient, the treatment is called brachytherapy (BT). Radioactiveatoms for use in the context of this invention can be selected from thegroup including, but not limited to, radium, cesium-137, iridium-192,americium-241, gold-198, cobalt-57, copper-67, technetium-99,iodine-123, iodine-131, and indium-111. Is also possible to label theantibody with such radioactive isotopes. Preferably the type IIanti-CD20 antibody and Bcl-2 inhibitor combination treatment is usedwithout such ionizing radiation.

Radiation therapy is a standard treatment for controlling unresectableor inoperable tumors and/or tumor metastases. Improved results have beenseen when radiation therapy has been combined with chemotherapy.Radiation therapy is based on the principle that high-dose radiationdelivered to a target area will result in the death of reproductivecells in both tumor and normal tissues. The radiation dosage regimen isgenerally defined in terms of radiation absorbed dose (Gy), time andfractionation, and must be carefully defined by the oncologist. Theamount of radiation a patient receives will depend on variousconsiderations, but the two most important are the location of the tumorin relation to other critical structures or organs of the body, and theextent to which the tumor has spread. A typical course of treatment fora patient undergoing radiation therapy will be a treatment schedule overa 1 to 6 week period, with a total dose of between 10 and 80 Gyadministered to the patient in a single daily fraction of about 1.8 to2.0 Gy, 5 days a week. In a preferred embodiment of this invention thereis synergy when tumors in human patients are treated with thecombination treatment of the invention and radiation. In other words,the inhibition of tumor growth by means of the agents comprising thecombination of the invention is enhanced when combined with radiation,optionally with additional chemotherapeutic or anticancer agents.Parameters of adjuvant radiation therapies are, for example, containedin WO 99/60023.

The type II anti-CD20 antibodies are administered to a patient accordingto known methods, by intravenous administration as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, or intrathecal routes. Intravenous or subcutaneousadministration of the antibodies is preferred.

The Bcl-2 inhibitors are administered to a patient according to knownmethods, e.g. by intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, or peroral routes. Intravenous, subcutaneous or oraladministration of the Bcl-2 inhibitors is preferred.

The invention also relates to a kit comprising a type II anti-CD20antibody and a selective Bcl-2 inhibitor for the combination treatmentof a patient suffering from a CD20 expressing cancer.

In an embodiment of the present invention, the kit further comprises apharmaceutically acceptable carrier. The kit may further include asterile diluent, which is preferably stored in a separate additionalcontainer. The kit may further include a package insert comprisingprinted instructions directing the use of the combined treatment as amethod for a CD20 expressing cancer disease, preferably a B-CellNon-Hodgkin's lymphoma (NHL).

The term “package insert” refers to instructions customarily included incommercial packages of therapeutic products, which may includeinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

In a preferred embodiment, the article of manufacture containers mayfurther include a pharmaceutically acceptable carrier. The article ofmanufacture may further include a sterile diluent, which is preferablystored in a separate additional container.

As used herein, a “pharmaceutically acceptable carrier” is intended toinclude any and all material compatible with pharmaceuticaladministration including solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and other materials and compounds compatible with pharmaceuticaladministration. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsof the invention is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

Pharmaceutical Compositions and Methods

Pharmaceutical compositions can be obtained by processing the type IIanti-CD20 antibody or the anti-Bcl-2 active agent according to thisinvention with pharmaceutically acceptable, inorganic or organiccarriers. Lactose, corn starch or derivatives thereof, talc, stearicacids or it's salts and the like can be used, for example, as suchcarriers for tablets, coated tablets, dragées and hard gelatinecapsules. Suitable carriers for soft gelatine capsules are, for example,vegetable oils, waxes, fats, semi-solid and liquid polyols and the like.Depending on the nature of the active substance no carriers are,however, usually required in the case of soft gelatine capsules.Suitable carriers for the production of solutions and syrups are, forexample, water, polyols, glycerol, vegetable oil and the like. Suitablecarriers for suppositories are, for example, natural or hardened oils,waxes, fats, semi-liquid or liquid polyols and the like.

The pharmaceutical compositions can, moreover, contain preservatives,solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners,colorants, flavorants, salts for varying the osmotic pressure, buffers,masking agents or antioxidants. They can also contain still othertherapeutically valuable substances.

Said pharmaceutical composition may further comprise one or morepharmaceutically acceptable carriers.

The present invention further provides a pharmaceutical composition, inparticular for use in cancer, comprising (i) an effective first amountof a type II anti-CD20 antibody, or (ii) an effective second amount of aselective Bcl-2 inhibitor. Such composition optionally comprisespharmaceutically acceptable carriers and/or excipients.

Pharmaceutical compositions of the type II anti-CD20 antibody alone usedin accordance with the present invention are prepared for storage bymixing an antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutical compositions of the anti-Bcl-2 active agent alone, e.g.the Bcl-2 inhibitor, depend on their pharmaceutical properties; e.g. forsmall chemical compounds such as e.g. ABT-737, ABT-199 or ABT-263, oneformulation could be e.g. the following:

a) Tablet Formulation (Wet Granulation):

Item Ingredients mg/tablet 1. Compound of formula (I) 5 25 100 500 2.Lactose Anhydrous DTG 125 105 30 150 3. Sta-Rx 1500 6 6 6 30 4.Microcrystalline Cellulose 30 30 30 150 5. Magnesium Stearate 1 1 1 1Total 167 167 167 831

Manufacturing Procedure:

1. Mix items 1, 2, 3 and 4 and granulate with purified water.2. Dry the granules at 50° C.3. Pass the granules through suitable milling equipment.4. Add item 5 and mix for three minutes; compress on a suitable press.

b) Capsule Formulation:

Item Ingredients mg/capsule 1. Compound of formula (I) 5 25 100 500 2.Hydrous Lactose 159 123 148 — 3. Corn Starch 25 35 40 70 4. Talc 10 1510 25 5. Magnesium Stearate 1 2 2 5 Total 200 200 300 600

Manufacturing Procedure:

1. Mix items 1, 2 and 3 in a suitable mixer for 30 minutes.2. Add items 4 and 5 and mix for 3 minutes.3. Fill into a suitable capsule.

In one further embodiment of the invention the pharmaceuticalcompositions according to the invention are two separate formulationsfor said type II anti-CD20 antibody and said Bcl-2 inhibitor.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interracialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition. Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The invention relates in part to a method for the treatment of a patientsuffering from cancer, particularly a CD20-expressing cancer, comprisingco-administering, to a patient in need of such treatment, a type IIanti-CD20 antibody and a selective Bcl-2 inhibitor. Said type IIanti-CD20 antibody and anti-Bcl-2 active agent are administered ineffective amounts.

In certain embodiments, a dosing cycle is for 28 days.

In certain embodiments of a method of treating cancer in a patient asprovided herein, the method comprises administering the type IIanti-CD20 antibody and the selective Bcl-2 for one or more dosing cyclesto the patient. In one embodiment, the one or more dosing cycles eachlast for at least one week. In another embodiment, the one or moredosing cycles are each for at least two weeks, three weeks, four weeks,five weeks, six weeks, seven weeks, eight weeks, nine weeks, or for morethan nine weeks. In one embodiment, each dosing cycle is four weeks.

In one embodiment, the therapeutic agents are administered to thepatient for one dosing cycle.

In another embodiment, therapeutic agents are administered to thepatient for more than one dosing cycle, for instance, for two, three,four, five, six, seven or more than seven dosing cycles. As an example,where a dosing cycle is four weeks, and the patient is administered withone or both therapeutic agents over six dosing cycles, the treatmentregimen will be for 24 weeks, such as illustrated in the dosing schemesshown in FIG. 3 .

In certain embodiments, methods are provided for the treatment of acancer in a patient in need thereof comprising administering to saidhuman a GA101 antibody and/or GDC-O199 in multiple dosing cycles.

In certain embodiments, the GA101 antibody and GDC-199 are bothadministered to the patient in one or more dosing cycles of the multipledosing cycles, and one of the GA101 antibody and GDC-0199 areadministered in one or more dosing cycles of the multiple dosing cycles.

In certain embodiments of the methods of treatment provided herein, thetherapeutic agents are administered to the patent in a dosing schemecomprising two or three treatment phases, where each treatment phasecomprises at least one dosing cycle that differs from the dosing cyclefrom other treatment phases. For example, in one embodiment where thedosing cycle comprises four weeks, the type II anti-CD20 antibody can beadministered to the patient once a week for two or more weeks of thefirst dosing cycle (e.g., a first treatment phase), and administeredonce per dosing cycle in the dosing cycles that follow the first dosingcycle (e.g., a second treatment phase).

In certain embodiments of the methods of treatment provided herein, thetype II anti-CD20 antibody is administered to the patient once a weekfor at least one week of a dosing cycle. In some embodiments, where thedosing cycle is for two or more weeks, the the type II anti-CD20antibody is administered to the patient once per dosing cycle.

In certain embodiments of the methods of treatment provided herein,GDC-0199 is administered once per day of a dosing cycle.

Both GDC-0199 and the type II anti-CD20 antibody can, for example, beadministered to the patient in a dosing cycle. In certain dosing cycles,one therapeutic agent alone is administered to the patient.

In certain embodiments of the methods provided wherein the therapeuticagents are administered to the patent in a dosing scheme comprisingmultiple dosing cycles, the multiple dosing cycles comprise a firsttreatment phase having dosing cycles wherein in each dosing cycle thetype II anti-CD20 antibody is administered once per dosing cycle andGDC-0199 is administered each day of the dosing cycle. Such dosingcycles in the first treatment phase can, for example, each be for fourweeks. In some embodiments, the multiple dosing cycles can furthercomprise a second treatment phase wherein the type II anti-CD20 antibodyalone is administered to the patient or wherein GDC-0199 alone isadministered to the patient (e.g., a maintenance phase).

In some embodiments of the methods provided herein wherein the type IIanti-CD20 antibody and GDC-0199 are both administered to the patient forone or more dosing cycles (e.g., in a treatment phase), the patient canthen be administered with GDC-0199 alone (e.g., in a maintenance phase).

In certain embodiments where GDC-0199 alone is administered to thepatient following combination therapy, GDC-0199 can, for example, beadministered once a day, once every other day, once every three days,four, five or six days, or once a week, to the patient.

In certain embodiments wherein the type II anti-CD20 antibody alone isadministered to the patient following combination therapy, the type IIanti-CD20 antibody can, for example, be administered once a week, onceevery two weeks or once per month, to the patient.

In certain embodiments of the methods of treatment provided herein, theamounts of GDC-0199 per dose administered to the patient are increasedduring a first dosing cycle. See, e.g., FIG. 2 for an exemplary dosingscheme where amounts of GDC-0199 administered to patients in the firstdosing cycle escalate from 50 mg doses the first week, to 100 mg dosesthe second week, to 300 mg doses the third week).

In certain embodiments, escalating doses of GDC-0199 are administered tothe patient prior to administration of the type II anti-CD20 antibody.In other embodiments, escalating doses of GDC-0199 are administered tothe patient after the type II anti-CD20 antibody has been administeredto the patient.

In some embodiments of the method of treatment provided herein, theamount of GDC-0199 administered to the patient per dose is increasedduring the first dosing cycle from initial amounts of between 10 mg to80 mg to final amounts of between 190 mg to 400 mg. In certainembodiments, the amount of GDC-0199 per dose administered to thepatients begins with 50 mg or 100 mg, and is increased to 300 mg perdose. In some embodiments, the amount of GDC-0199 in the initial dosesadministered to the patient can, for example, be between 20 mg to 60 mg(e.g., 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mgdoses), followed by dose amounts of 100 mg, 200 mg, 300 mg or more ofGDC-0199.

In certain embodiments of the methods provided herein, doses of GDC-0199are administered to the patient in increasing amounts prior to the firstadministration of the type II anti-CD20 antibody. In some embodiments,doses of GDC-0199 are administered to the patient in increasing amountsafter the first administration of the type II anti-CD20 antibody.

As used herein, the term “patient” typically refers to a human in needof treatment with type II anti-CD20 antibody (e.g. a patient sufferingfrom CD20 expressing cancer) for any purpose, and, in one embodiment, ahuman in need of such a treatment to treat cancer, or a precancerouscondition or lesion. However, the term “patient” can also refer tonon-human animals, preferably mammals such as dogs, cats, horses, cows,pigs, sheep and non-human primates, among others.

The invention further comprises a type II anti-CD20 antibody for thetreatment of CD20 expressing cancer in combination with a selectiveBcl-2 inhibitor.

The invention further comprises a type II anti-CD20 antibody for thetreatment of a patient suffering from a CD20 expressing cancer incombination a selective Bcl-2 inhibitor.

The invention further comprises a type II anti-CD20 antibody and aselective Bcl-2 inhibitor for use in the treatment of CD20 expressingcancer.

The invention further comprises a type II anti-CD20 antibody and aselective Bcl-2 inhibitor for use in the treatment of a patientsuffering from a CD20 expressing cancer.

In one embodiment, said selective Bcl-2 inhibitor is ABT-199.

In one embodiment, said type II anti-CD20 antibody has a ratio of thebinding capacities to CD20 on Raji cells (ATCC-No. CCL-86) of said typeII anti-CD20 antibody compared to rituximab of 0.3 to 0.6, and in oneembodiment, 0.35 to 0.55, and in another embodiment, 0.4 to 0.5.

In one embodiment, said type II anti-CD20 antibody is a GA101 antibody.

In one embodiment, said type II anti-CD20 antibody has increasedantibody dependent cellular cytotoxicity (ADCC).

In certain embodiments of the methods of treatment of a cancer in apatient provided herein, the cancer is a non-solid tumor. In oneembodiment, the non-solid tumor is a CD20 expressing non-solid tumor.Exemplary non-solid tumors that can be treated in the methods providedherein, include, for instance, a leukemia or a lymphoma. In oneembodiment, the non-solid tumor is a B cell lymphoma.

In one embodiment, the CD20 expressing cancer is a B-Cell Non-Hodgkin'slymphoma (NHL).

In one embodiment, said type II anti-CD20 antibody is a monoclonalantibody.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

EXAMPLES Example 1. Treating Lymphoma with a Combination of GDC-0199 andObinutuzumab

Nonclinical data supports the hypothesis that the combination ofGDC-0199 and GA101 (in this case, obinutuzumab) will show moreanti-tumor activity than when each drug is administered alone. The studyused a non-Hodgkin's lymphoma (NHL) xenograft model of aggressivelymphoma, the diffuse large B-cell lymphoma (DLBCL) derived cell lineSU.DHL-4. Obinutuzumab was administered at a dose of 1 mg/kg IV, once aweek for 3 weeks and achieved tumor stasis followed by growth delay.GDC-0199 was administered at 100 mg/kg QD for 21 days and alsodemonstrated stasis followed by tumor growth delay. However, thecombination of GDC-0199 and obinutuzumab induced a greater than additiveeffect resulting in tumor regressions (5 of 8 partial regressions (PRs);see FIG. 1 ). Three weeks of combination therapy resulted in enhancedtumor growth inhibition (TGI) (118% TGI), compared with 76% (GA101) and80% (GDC 0199) TGI observed with single agent administration (see FIG. 1). Increased tumor regressions (5 PRs) were also observed when GA101 wascombined with GDC 0199, compared with single agent administration.Additionally. TGI was sustained in the combination treatment group aftertreatment ended on day 21 since 116% TGI was observed at day 31 (10 daysafter dosing ended), versus 30% TGI with GA101 and 25% TGI with GDC 0199as single agents. In summary, GA101 in combination with GDC 0199resulted in increased TGI and tumor regressions compared to each agentadministered separately in a NHL xenograft model.

In this study the vehicles for GA-101 and GDC-0199 were saline and 60%phosal, respectively. In FIG. 1 , the dose of GDC-0199 is expressed asfree base equivalents in mg/kg of body weight. Results are expressed asthe fitted tumor volume as determined by linear mixed effects modelingfor each treatment group vs. time in days, where Day 0 is the first dayof treatment.

Example 2. A Phase Ib Multicenter Study of GDC-0199 and Obinutuzumab inPatients with Relapsed or Refractory or Previously Untreated ChronicLymphocytic Leukemia

Two schedules will be evaluated: Schedule A (FIG. 2 ), consisting ofGDC-0199 administered in escalating doses for 3 weeks prior to the firstobinutuzumab infusion, and Schedule B (FIG. 3 ), consisting ofobinutuzumab administered first followed by escalating dose levels ofGDC-0199. Schedule A and Cohort 1 of Schedule B will be enrolled inparallel. In addition, the dose-finding stage will evaluate whether theadministration of GDC-0199 prior to the first obinutuzumab infusion(Schedule A) will result in a lower incidence of infusion reactions,thereby reducing the need for split doses of obinutuzumab andcorticosteroid premedication.

The expansion stage will include two expansion cohorts of 20 patientseach (relapsed/refractory and previously untreated CLL) and willevaluate the safety and preliminary efficacy of the selected combinationdose and schedule.

In Schedule A, the combination treatment of GDC-0199 and obinutuzumabwill be administered for a total of 7 cycles of 28 days each, includinga total of 8 infusions of obinutuzumab and GDC-0199 QD.

In Schedule B, the combination treatment of GDC-0199 and obinutuzumabwill be administered for a total of 6 cycles of 28 days each, includinga total of 9 infusions of obinutuzumab (8 doses; first dose will besplit into two infusions) and GDC-0199 QD.

GDC-O199 monotherapy may be continued in patients beyond 6-7 cycles ofcombination treatment described above (e.g., if they have acceptabletoxicity and have not yet achieved maximal clinical response (i.e., arehaving continued improvement/reduction in tumor burden that has not yetstabilized for at least 2 months)). Such patients may continue GDC-0199monotherapy until they have achieved maximal response or up to 1 yearafter the last patient is enrolled, whichever occurs first.

Example 3. Antitumor Activity of Combined Treatment of GDC-0199 and TypeII Anti-CD20 Antibody (Obinutuzumab) as Compared to Combined Treatmentof GDC-0199 and a Type I Anti-CD20 Antibody (Rituximab)

Test agents. The type II anti-CD20 antibody was the GA101 antibody IgG1(a chimeric humanized IgG1 antibody as disclosed in WO 2005/044859(termed B-HH6-B-KV1 GE therein, also known as obinutuzumab orRO5072759), which was provided as stock solution (conc. 9.4 mg/ml) fromRoche GlycArt, Schlieren, Switzerland. Antibody buffer includedhistidine, trehalose and polysorbate 20. Antibody solution was dilutedappropriately in PBS from stock for prior injections. GDC-0199 wasobtained from Genentech Inc., CA, USA.

Cell line and culture conditions. The human Z138 mantle cell lymphomacell line is routinely cultured in DMEM supplemented with 10% fetalbovine serum (PAA Laboratories, Austria) and 2 mM L-glutamine at 37° C.in a water-saturated atmosphere at 5% CO₂. Cells were co-injected withMATRIGEL.

Animals. Female SCID beige mice; age 5-6 weeks at arrival (purchasedfrom Charles River. Sulzfeld, Germany) were maintained underspecific-pathogen-free condition with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).Experimental study protocol was reviewed and approved by localgovernment (Regierung von Oberbayern; registration no.55.2-1-54-2531.2-26-09). After arrival animals were maintained in thequarantine part of the animal facility for one week to get accustomed tonew environment and for observation. Continuous health monitoring wascarried out on regular basis. Diet food (Altromin Spezialfutter GmbH &Co. KG) and water (filtered) were provided ad libitum.

Monitoring. Animals were controlled daily for clinical symptoms anddetection of adverse effects. For monitoring throughout the experimentbody weight of animals was documented two times weekly and tumor volumewas measured by caliper after staging.

Treatment of animals. Animal treatment was started at the day ofrandomisation 18 days after tumor cell inoculation. RO5072759 orrituximab were administered as single agent, i.p., once weekly (day 18,25, 32) for 3 weeks at dosages of 1 mg/kg. The corresponding vehicle wasadministered on the same days. GCD-0199 was given p.o. once daily (fromday 18 to day 34), over 17 days at a dosage of 100 mg/kg. In thecombination therapy groups, the antibodies and GDC-0199 wereadministered at the same dosages and on the same days.

Tumor growth inhibition study in vivo. Results of therapy on tumorvolume development are shown in FIG. 4 . On day 35 after tumor cellinoculation, there was a tumor growth inhibition of 32%, 59%, 73%, 96%or 106% (regression) in the animals given rituximab, GDC-0199,RO5072759, combination of GDC-0199 plus rituximab or combination ofGDC-0199 plus RO5072759, respectively, compared to the control group.

Example 4. Administration of GDC-0199 as a Single Agent FollowingCombination with Obinutuzumab Results in Significant Delay in TumorRegrowth

This example describes results using the DLBCL SU-DH L-4 xenograftmodel, which discussed in Example 1 above. Initially, GDC-0199 was dosedorally for 21 continuous days in combination with GA101 (in thisexample, obinutuzumab) at 1 mg/kg for 3 weeks. The latter resulted inenhanced TGI (91%), compared with 54% (GA101) and 24% (GDC-0199) TGIobserved with each agent alone (FIG. 5 ). At day 22 tumor bearing micein the combination cohort continued to be dosed with GDC-0199 alone at100 mg/kg for an additional 24 days. The latter resulted in asignificant delay in tumor regrowth when compared to mice treated withthe combination of GA101 and GDC-0199 over a 21 day period (time totumor progression of the combination cohort=38 days vs. continuedtreatment with GDC-0199=45 days (FIG. 5 ). Thus, single-agent treatmentwith GDC-0199 following combination with GA101 sustains efficacy invivo. These results support a benefit for maintenance therapy withGDC-0199.

In FIG. 5 , the control is saline vehicle for GA101 plus 60% phosalvehicle for GDC-0199. GA101 was dosed intravenously once a week (QW) for3 weeks and GDC-0199 was dosed orally and daily (QD) for 21 days (QD×21)as single agents or in combination. As explained above, a cohort oftumor bearing mice was also dosed with GDC-0199 alone for an additional24 days after combination treatment ended on day 21 (OD×45). Under thex-axis, treatment periods are denoted by solid black lines (

) for the combination cohorts while the continued single agent GDC-0199treatment is denoted by dashed black line (

).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

What is claimed is:
 1. A method for the treatment of a cancer in a humanin need thereof comprising administering to said human an effectiveamount of a GA101 antibody or2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods, followed by co-administering an effective amount of said GA101antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods.
 2. A method for the treatment of a cancer in a human in needthereof comprising administering to said human an effective amount of aGA101 antibody or2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 days, followed by co-administering aneffective amount of said GA101 antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof for one or more dosingperiods.
 3. The method of claim 1, wherein an effective amount of saidGA101 antibody is administered once every dosing period for 1, 2, 3, 4,5 or 6 cycles, followed by co-administrating the effective amount ofsaid GA101 antibody antibody once every dosing period and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof one, two or three times aday for one or more dosing periods.
 4. The method of claim 1, wherein aneffective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is administered one, twoor three times a day for 1, 2, 3, 4, 5 or 6 dosing periods, followed byco-administering an effective amount of said GA101 antibody once everydosing period and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof one, two or three times aday for one or more dosing periods.
 5. The method of claim 4, whereinthe effective amount of said GA101 antibody is from about 500 mg toabout 3000 mg and the effective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is from about 20 mg toabout 500 mg.
 6. The method of claim 4, wherein the effective amount ofsaid GA101 antibody is 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500mg, and the effective amount of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, or 300 mg.
 7. The method of claim 1,wherein said GA101 antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof are co-administeredsequentially during each dosing period, and each dosing period is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.
 8. The method of claim1, wherein said GA101 antibody is an anti-human CD20 antibody comprisingan HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, an HVR-H2comprising the amino acid sequence of SEQ ID NO:2, an HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:3, an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:4, an HVR-L2 comprising the amino acidsequence of SEQ ID NO:5, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:6.
 9. The method of claim 8, wherein said GA101antibody further comprises a V H domain comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising the amino acidsequence of SEQ ID NO:8.
 10. The method of claim 1, wherein said GA101antibody comprises an amino acid sequence of SEQ ID NO:9 and an aminoacid sequence of SEQ ID NO:
 10. 11. The method of claim 1, wherein inthe GA101 antibody is known as obinutuzumab.
 12. The method of claim 1,wherein the GA101 antibody comprises an amino acid sequence that has atleast 95% sequence identity with amino acid sequence of SEQ ID NO:9 andat least 95% sequence identity with an amino acid sequence of SEQ ID NO:10.
 13. The method of claim 1, wherein the cancer is a CD20—expressingcancer.
 14. The method of claim 13, wherein the cancer is a non-solidtumor.
 15. The method of claim 13, wherein the cancer is a lymphoma or aleukemia.
 16. The method of claim 13, wherein the leukemia is chroniclymphocytic leukemia (CLL).
 17. The method of claim 16, wherein thepatient is suffering from relapsed or refractory or previously untreatedchronic lymphocytic leukemia.
 18. A method for the treatment of a cancerin a human in need thereof comprising co-administering to said human aneffective amount of a GA101 antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamidein a dosing period, wherein the GA101 antibody is administered at500-3000 mg weekly and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideis administered at 50-300 mg 1-3 times per day in the dosing period. 19.A method for the treatment of a cancer in a human in need thereofcomprising administering to said human a GA101 antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-21H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamidein multiple dosing cycles, wherein each dosing cycle is for at least 2,3, 4, 5 or 6 weeks, and 500 mg to 3000 mg of the GA101 antibody isadministered once per dosing cycle for one or more dosing cycles of themultiple dosing cycles, and 10 mg to 300 mg of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideis administered each day per dosing cycle for one or more dosing cyclesof the multiple dosing cycles.
 20. The method of claim 19, wherein boththe GA101 antibody and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideare administered to the patient in at least 2, 3, 4, 5, 6, 7, 8, or morethan 8, dosing cycles of the multiple dosing cycles.
 21. The method ofclaim 19, wherein following the last dosing cycle of the multiple dosingcycles, doses of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideare administered to the patient in the absence of the GA101 antibodybeing administered to the patient.
 22. The method of claim 21, whereinthe doses of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideadministered to the patient in the absence of the GA101 antibody arebetween about 10 mg to about 300 mg2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamide.23. The method of claim 22, wherein the the doses of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamide administered to the patient in the absence of the GA101antibody are administered to the patient for at least 3, 4, 5, 6, 7, 8days, or for 10 or more days, 20 or more days, or 30 or more days. 24.The method of claim 19, wherein the multiple dosing cycles comprise anescalating dosing cycle in which2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenylsulfonyl)benzamideis administered to the patient in escalating daily dose amounts duringthe escalating dosing cycle.
 25. The method of claim 24, wherein theescalating daily dose amounts comprise an initial daily dose amount of10 mg and a final daily dose amount of 300 mg.
 26. The method of claim19, wherein the cancer is a non-solid tumor.
 27. The method of claim 19,wherein the cancer is chronic lymphocytic leukemia (CLL).
 28. The methodof claim 13, wherein the cancer is non-Hodgkin's lymphoma (NHL).
 29. Themethod of claim 13, wherein the cancer is acute myeloid leukemia (AML).